1. Field of the Invention
The present invention relates generally to immunosuppression and the use of cyclosporin. More specifically, the present invention relates to a method of evaluating an immunosuppressive regimen on a patient-by-patient basis.
2. Description of the Related Art
The addition of cyclosporine (CSA) to the immunosuppressive regimens following organ transplantation has resulted in a marked improvement in graft survival (1,2). Cyclosporine, which remains an important component of most anti-rejection immunosuppressive regimens is, however, a problematic drug to use. The optimal clinical use of cyclosporine aims to both maximize efficacy, thus maintaining graft survival and to minimize drug-related toxicity.
Optimal clinical use has remained controversial, not only because of a poorly defined relationship between the dose of cyclosporine administered and the concentration of cyclosporine achieved, but also because both cyclosporine dose and concentration are poor predictors of clinical efficacy and toxicity (3-6). Thus, as detailed in recent reviews (4-6), numerous studies have examined the relationship between clinical efficacy and various pharmacokinetic parameters of cyclosporine disposition, such as trough concentrations, area-under-the-concentration-time curve (AUC), average cyclosporine concentrations and clearance. Some studies have shown a relationship between each of these pharmacokinetic measures and efficacy, but the correlation between any measure of cyclosporine disposition and efficacy is poor. This is reflected by a wide range of clinical practice, as demonstrated in the most recent consensus guidelines on the monitoring of cyclosporine concentrations after organ transplantation, where a wide range of cyclosporine trough concentrations was designated as "therapeutic" by different institutions (7).
The pharmacokinetics of cyclosporine are highly variable, both between patients and within the same patient (3). Even with the new microemulsion preparation of cyclosporine, which has less inter-individual and intra-individual pharmacokinetic variability than the older formulation pharmacokinetic characteristics, cyclosporine may vary 2-3 fold among individuals and 1-2 fold in the same individual (8-10). This pharmacokinetic variability has resulted in much of the effort being focused on the measurement of drug concentrations, and subsequent manipulation of dose, in order to achieve target cyclosporine concentrations within a poorly defined "therapeutic range" (11). However, it has long been noted that clinical response, with regard to both efficacy and drug-related toxicity, may be different in patients with similar concentrations of cyclosporine (5,12). This suggests that, in addition to interindividual pharmacokinetic differences in drug disposition, interindividual pharmacodynamic differences in response to drug may contribute to the weak relationship between concentrations of cyclosporine and clinical response.
The immunosuppressive action of cyclosporine may be mediated largely through effects on lymphocytes. After intracellular binding to cyclophilin, the cyclosporine-cyclophilin complex binds and inhibits the action of calcineurin, thereby reducing nuclear translocation of the cytoplasmic subunit of the nuclear factor of activated T-cells to the nuclear subunit, resulting in decreased T-cell receptor transcription of the interleukin-2 (IL-2) gene (13,22). Inhibition of IL-2 production is thought to be critical to the immunosuppressive effect of cyclosporine (13) and the inhibitory effect of cyclosporine on lymphocyte proliferation can be reversed by the addition of exogenous IL-2 (13). Further evidence for the immunological importance of inhibition of IL-2 production is provided by recent studies showing that blockage with monoclonal antibodies of the IL-2 receptor reduced the frequency of transplant rejection (23).
The inhibition of mitogen stimulated IL-2 in isolated peripheral blood lymphocytes by cyclosporine has been studied as a potential pharmacodynamic measure of the effect of cyclosporine (18,24,25). Such studies have generally shown that IL-2 production is inhibited 35-40% in patients receiving cyclosporine (18). However, the cumbersome technique, which required isolation of lymphocytes, culture with mitogen for 48 hours and measurement of IL-2 by bioassay, as well as the highly variable interindividual responses and the lack of a well-defined association with cyclosporine concentrations achieved in vivo, have limited the practical application of this technique. Nevertheless, the potential importance of using IL-2 inhibition as a marker of the effect of cyclosporine was demonstrated by the observation that failure to inhibit IL-2 production was associated with an increased likelihood of organ rejection (16). These studies therefore suggested that a more refined measurement of cyclosporine-induced inhibition of IL-2 production might define a biologically relevant effect that would thus allow development of a pharmacodynamic measure to determine interindividual variability in response to cyclosporine.
The inhibitory effect of cyclosporine on interleukin-2 (IL-2) production is thought to be critical to its immunosuppressive action (13). Several investigators have attempted to use the inhibitory effect of cyclosporine on IL-2 production, or on IL-2-dependent lymphocyte proliferation, as pharmacodynamic measures of response to cyclosporine (14-18). However, these techniques have been problematic. Plasma concentrations of IL-2 in humans are low, and it is therefore not practical to reliably detect a cyclosporine-induced, concentration-dependent, decrement of plasma IL-2 concentrations (14). Studies have thus examined either the effect of plasma from patients receiving cyclosporine on the proliferation of normal third party lymphocytes, or have otherwise isolated lymphocytes from patients who have received cyclosporine and examined the ability of these isolated lymphocytes to proliferate in culture or to produce IL-2 after mitogen stimulation.
Plasma obtained from patients receiving cyclosporine is not ideal for determining a pharmacodynamic response since approximately 50-70% of the drug is concentrated in erythrocytes in a temperature-dependent fashion (5,19). Thus, plasma cyclosporine concentrations are much lower than whole blood cyclosporine concentrations, and are critically dependent on the temperature at which the plasma is separated from blood. Plasma is therefore an unsuitable matrix for the measurement of both the pharmacokinetic and pharmacodynamic characteristics of cyclosporine. For these reasons, determination of cyclosporine concentrations in patients for therapeutic monitoring is now almost exclusively performed in whole blood samples rather than in plasma (7).
The alternative strategy of using isolated lymphocytes in culture (ex vivo) to measure a pharmacodynamic effect of cyclosporine is also problematic. First, the isolation of lymphocytes from blood separates these lymphocytes from the complex, cyclosporine-rich milieu in which their pharmacodynamic response occurs. This may alter both the response and rate of recovery (20). Second, isolation of lymphocytes involves multiple washes, which are likely to variably deplete cyclosporine concentration within the isolated lymphocytes. Third, the requirement of lymphocytic cell culture for 48 hours or longer limits the practical application of any pharmacodynamic measure obtained using such techniques.
The lack of a biologically relevant, practical pharmacodynamic measure of the effect of cyclosporine has been a significant obstacle to defining interindividual variability in response to cyclosporine, and development of such a measure has remained a n elusive goal (4,5). Thus, the prior art is deficient in a method to evaluate the biological effects of drugs such as cyclosporine for individual patients, i.e., an evaluation which would provide more information than mere measurement of drug concentration. The present invention fulfills this long-standing need and desire in the art.